Monocytes are blood borne cells essential for effective innate and adaptive host defenses against infections with intracellular bacterial pathogens. Our laboratory and others identified sub-populations of blood monocytes in the mouse capable of functioning differentially as inflammatory or homeostatic cells. Remarkably, we found the same inflammatory monocytes that are recruited to infected tissues to kill Listeria monocytogenes (Lm) also are parasitized and transport intracellular Lm into the brain. Understanding why inflammatory monocytes that eventually become cytokine-activated effector cells are susceptible to intracellular parasitism is essential because most category A and B bacteria on the NIAID priority list are intracellular pathogens. The objective of this application is to establish the biological basis for parasitism of inflammatory monocytes during systemic infection with Lm, an intracellular pathogen relevant to biodefense. Our central hypothesis is that developmental immaturity prevents bone marrow monocytes and their precursors from responding optimally to cytokine activation to kill intracellular bacteria so that these cells are targeted for infection in the bone marrow then enter the circulation as infected monocytes. This hypothesis is based on our analysis of mouse monocyte sub-populations during systemic Lm infection and preliminary data showing parasitism of EP-MP12+CD11b+Ly-6Chi mononuclear cells in the bone marrow. This hypothesis will be tested with the following aims; 1) To elucidate the cellular basis of bone marrow parasitism during systemic Lm infection, 2) To elucidate why inflammatory monocytes are susceptible to parasitism by Lm, and 3) To determine from which organ infected monocytes enter the bloodstream. These aims will be accomplished using the well-established mouse model of Lm infection. This research is innovative because it applies the newly-recognized paradigm of monocyte heterogeneity in the mouse, to an established animal model of infection with an intracellular bacterium. Since human correlates of mouse monocyte sub-populations have been identified, our results will allow a better understanding than previously possible of how human monocytes function during the innate response to systemic infection. We expect that successful completion of these aims will reveal bone marrow to be an under-explored, but critical target organ for Lm , infection in vivo, and is the organ in which inflammatory monocytes are parasitized then emerge into the blood as Trojan horses. This will enable us to make clear key pathways of immunological activation of inflammatory monocytes manifested in vivo for killing intracellular bacteria in sub-lethal infection, but which are inadequate or dysfunctional during lethal disease. These data will then catalyze development of rational therapies to improve host defenses against severe and overwhelming infection by category A and B intracellular bacterial pathogens.